Cancer of the cervix is the most common cancer in women (.about.25% of all female cancer). Moreover, the incidence is increasing in younger women. Indeed, approximately 2% of routine cervical smears show abnormal cytology, indicating an epidemic. Such an epidemic is current in many western and developing countries. Sexual activity appears to be an important predisposing factor in the epidemiology of cardinogenesis and precancerous lesions. An early age of sexual intercourse and multiplicity of sexual partners are associated statistically with an increased risk of malignancy [Harris et al., Br. J. Cancer 42: 359-63, 1980]. The consorts are often men with penile warts ("high risk males"), and a very high proportion (&gt;90%) of cervical carcinoma tissue contain detectable DNA sequences for known varieties of the human papillomavirus (HPV). This supports a growing body of evidence implicating certain types of HPV as the sexually transmitted factor involved in the development of squamous-cell carcinoma of the cervix [zur Hausen et al., Progr. Med. Virol. 30: 170-86, 1984; zur Hausen, Prog. Med. Virol. 32: 15-21, 1985; zur Hausen, Cancer 59: 1692-6; Campion et al., Lancet 1: 943-6, 1985]. The prevalence of cervical cancer and precancerous lesions is becoming increasingly more common in younger women. Without treatment it can be fatal, the death rate being .about.100 per million women per year in Western countries. Fortunately, if detected at an early stage, effective treatment is available that can eliminate the fatal consequences.
The immediate management and subsequent follow-up of young women with abnormal cytological smears who still wish to bear children presents many problems. This has been compounded by uncertainty about the interpretation of smears with features of papillomavirus infection ("kollocytes") as well as dysplasia. Moreover, the current cytological testing tool for cervical cancer screening, the Pap smear, has a false negative rate of .about.20%. Significant numbers of dysplastic lesions regress spontaneously, others fail to progress, while a few progress rapidly. Thus, from an ill-defined cloud of morphological abnormalities occasional cancers develop. At present there is no clear way to predict whether cancer will result if a Pap smear happens to be abnormal. Clinical examination of many of these patients has failed to find warty lesions (condylomata accuminata) on the external genitalia or, indeed, on the cervix itself. The more difficult procedure of colposcopy, after the application of 3% acetic acid, is, in fact, required, revealing the presence of flat ("non-condylomatous") warts (which are invisible to the naked eye). These are the expected premalignant lesions. Histophathological progression of the wart to carcinoma in situ and frank malignancy has been well described [e.g., Dyson et al., J. Clin. Path. 37: 126-31, 1984]. An increasingly prevalent problem is the occurrence of invasive cancer within 3 years of a negative Pap smear [Berkowitz et al., Gynecol. Oncol. 8: 311, 1979; Holman et al., Med. J. Aust. 2: 597, 1981]. Whereas the presence of papillomavirus replication may be confirmed in cervical condylomata by detection of virus particles or the group-specific antigen, neither particles nor antigen have, however, been found in squamous cell carcinoma tissue.
In contrast to the uncertainty and controversy that surrounds the interpretation of tests based on morphology, the new techniques in molecular biology can be utilised to bypass such problems and provide more objective information. By using nucleic acid hybridization techniques the viral DNA can be identified directly and at an earlier stage of infection. Indeed, using these approaches, HPV types have been found in both benign and premalignant lesions.
At present .about.50 types of the papillomavirus have been distinguished in human infection. Different ones infect different epithelial areas. The particular types of HPV that commonly infect the genital tract include those assigned the numbers 6, 11, 16, 18 and several rarer types (31, 33, 35, 39, 43 and 44). HPV6 [de Villlers et al., J. Virol. 40: 932-5, 1981] and HPV11 [Gissmann et al., J. Virol. 44: 393-400, 1982; Gissman et al., Proc. Natl. Acad. Sci. USA 80: 560-3, 1983; Dartmann et al., Virology 151: 124-30, 1986] have been associated with benign condylomata accuminata, the classical lesion of the anal and genital tract [Gissmann et al., J. Invest. Dermatol. 83: 26s-8s, 1984]. In contrast, HPV16 [Durst et al., Proc. Natl. Acad. Sci. USA 80: 3812-5, 1983] and HPV18 [Boshart et al., EMBO J. 3: 1151-7, 1984; Cole and Danos, J. Mol. Biol. 193: 599-608, 1987] are more often detected in dysplastic flat lesions of the vulva and cervix, and squamous carcinoma of the cervix and penis [Crum et al., Cancer 49: 468-71, 1982; Campion et al., Lancet i: 943-6, 1985].
Thus the types of HPV that infect the anogenital area can be assigned to two categories as follows:
1. "Low-risk"; HPV 6 and HPV11, with type 6 being the most common of all anogenital types. PA0 2. "High-risk"; HPV16, HPV18, HPV31, HPV33, HPV35, HPV39 HPV43 and HPV44. PA0 The sequence of HPV6b is given in Schwartz et al., EMBO J. 2: 2341-2348. PA0 The sequence of HPV11 is given in Dartmann et al., Virology 151: 124-30, 1986. PA0 The sequence of HPV16 is given in Seedort et al., Virology 145: 181-5, 1985. PA0 The sequence of HPV18 is given in Matlashewski et al., J. Gen. Virol. 67: 1909-16, 1986. PA0 The sequence of HPV33 is given in Cole and Streeck, J. Virol. 58: 991-5, 1986.
The frequency of occurence of the higher risk types is in decreasing order. Thus, within the high risk category, HPV16 is most common (45-60%), HPV18 is next most common (20-30%) and the others are rarer, the last 4 being discovered only recently and reported in 1986 (total frequency for all of these rarer types is, collectively, .about.15%). Other rarer types are likely to be discovered in due course.
In support of a role for HPVs in cervical cancer the following findings are noteworthy:
(i) DNAs of known high risk HPVs have been detected in .about.90% of cervical adenocarcinomas and squamous cell carcinomas [Zachow et al., Nature 300: 771-3, 1982; Gissmann et al., 1984, ibid].
(ii) High risk HPV DNA has been found in metastases arising from cervical tumours [Lancaster et al., Am. J. Obstet. Gynecol. 154: 115-9, 1986].
(iii) Instead of being present in cells in the usual episomal form, DNAs of high risk HPVs have been found integrated into human genomic DNA [Schwartz et al., Nature 314: 111-4, 1985; Lehn et al., Proc. Natl. Acad. Sci. USA 82: 5540-4, 1985; Kreider et al., Nature 317: 639-41, 1985; Matsukura et al., J. Virol. 58: 979-82, 1986; Schneider-Gadicke and Schwartz, EMBO J. 5: 2285-92, 1986; Di Luca et al., J. Gen. Virol. 67: 583-9, 1986]. Such integration has been suggested to be necessary for malignant conversion of the cells, supported by findings of integration also in precarcinoma tissue [Shirasawa et al., J. Gen. Virol. 67: 2011-5, 1986].
(iv) The integration pattern usually interrupts or deletes specific regions of the HPV16 or 18 DNA, but consistently leaves intact the E6 and E7 openreading frames (ORFs) ([Pater and Pater, Virology 145: 313-8, 1985], which continue to express, at least in cell lines derived from cervical carcinomas [Smotkin and Wettstein, Proc. Natl Acad. Sci. USA 83: 4680-4, 1986; Androphy et al., EMBO J. 6: 989-92, 1987; Baker et al., J. Virol. 61: 962-71, 1987; Takebe et al., Biochem. Biophys. Res. Commun. 143: 837-44, 1987].
(v) A splice donor exists in the E6 ORF of HPV16 and 18 (but not HPV6 and 11) which can result in the generation of an ORF which when translated resembles epidermal growth factor [zur Hausen, Lancet 489-91, 1986].
(vi) Integration in cervical cell lines (HeLa, CaSki, SiHa, SW756, etc) is often near proto-oncogenes [Durst et al., Proc. Natl. Acad. Sci. USA 84: 1070-4, 1987; Popescu et al., Cytogenet. Cell Genet, 44: 58-62, 1987; Popescu et al., J. Virol, 51: 1682-5, 1987; Shirasawa et al., J. Gen. Virol. 68: 583-91, 1987].
(vii) Such integration is associated with increased expression of c-myc and c-ras mRNA [Durst et al., 1987, ibid; Shirasawa et al., 1987, ibid.), consistent with the suggestion that cis-activation of cellular oncogenes by HPV might be associated with malignant transformation of cervical cells.
(viii) Human fibroblasts and keritanocytes can be transformed by transfection with HPV16 [Pirisi et al., J. Virol. 61: 1061-6, 1987], as can NIH 3T3 cells [Tsunokawa et al., Proc. Natl. Acad. Sci. USA 83: 2200-3, 1986; Yasumoto et al., J. Virol. 57: 572-7, 1986].
(ix) Integration might disrupt genes coding for cellular interfering factors: this may cripple the cells normal defense mechanism that suppresses uncontrolled growth and transcription of the virus [zur Hausen, Lancet 489-91, 1986].
Whereas penile warts in males only very rarely result in cancer of the penis, when transmitted to the cervix cancer is much more likely to follow. About one-third of patients who have histologically-confirmed HPV infection of the cervix can be expected to develop cervical intraepithelial neoplasia (CIN) within a year [Nashet al., Obstet. Gynecol. 69: 160-2, 1987]. The lag time between infection and cancer is, however, often 10-30 years. Thus the unique environment of the cervix, coupled with other factors, such as smoking [Trevathan et al., J. Am. Med. Ass. 250: 449-504, 1983], also contributed to the onset of the cancer. Damage of DNA by the latter, coupled with HPV's action to cause cellular proliferation, may explain the onset of malignancy. Treatment of women with precancerous lesions involves surgical extirpation of the affected area. Moreover, it is now believed by many that treatment should also involve her infected male consort, in order to avoid reinfection and infection of other women by the man.
Since the cytological test used presently for routine screening of cervical cells (Pap smear) is considered subjective and does not allow the identification of the particular type of HPV in a lesion, we foresee that the more specific approach of DNA--DNA hybridization for direct viral detection will in due course be used routinely to supplement or even replace cytology in primary screening.
In the clinical evaluation of a patient it is important to distinguish between those lesions harbouring the potentially carcinogenic (high-risk) types from those associated with the more benign (low-risk) types.
The evaluation of patient specimens for HPV infection has been facilitated by techniques of filter hybridization. Such techniques have been used to detect HPV DNA in cervical scrapes collected in parallel with samples for routine cytology [Wagner et al., Obstet. Gynecol. 64: 767-72, 1984; Wickenden et al., Lancet i: 65-7, 1984; Schneider et al., Science 216: 1065-70, 1982]. In January 1985 a project was begun by Dr. Morris at the University of Sydney to develop a direct test for the anogenital types of HPV; with a particular aim of evaluating whether the infection was by one of the high risk types of HPV or by one of the low risk types. The first publication describing a preliminary version of the test, which involved recombinant viral DNAs, is: B. R. Henderson, C. H. Thompson, B. R. Rose, Y. E. Cossart and B. J. Morris, "Detection of specific types of human papillomavirus in cervical scrapes, anal scrapes, and anogenital biopsies by DNA hybridization", Journal of Medical Virology 12: 381-93, 1987. This paper was submitted early in 1986. Since then the sensitivity of the recombinant DNA test has been increased 100-fold and further improvements are being made continually. Over 5,000 clinical specimens have been tested to date. These have been mainly from Sydney S.T.D. clinics. The data so far has established the viability and usefulness of direct viral detection for determination of the presence and nature of HPV infection in cervical scrapes and other anogenital specimens. Our other recent publications using this approach include: B. R. Rose, C. H. Thompson, A. M. McDonald, B. R. Henderson, Y. E. Cossart & B. J. Morris, "Cell Biology of cultures of anogenital warts", British Journal of Dermatology 116: 331-22, 1987; B. J. Parker, Y. E. Cossart, C. H. Thompson, B. R. Rose & B. R. Henderson, "The clinical management and laboratory assessment of anal warts", Medical Journal of Australia 147: 59-63, 1987; P. M. Katelaris, Y. E. Cossart, B. R. Rose, B. Nightingale, E. Sorich, C. H. Thompson, P. B. Dallas & B. J. Morris, "Human papillomavirus: The untreated male reservoir", Journal of Urology, in press, 1988. Much other work has yet to be published.
For main strand DNA sequences of the most common anogenital HPV types see as follows:
Principal of detection of specific viral DNA by hybridization
DNA is double-stranded. Each strand of DNA is a complementary `mirror image` of the other. The DNA strands are held together by hydrogen bonding. Our techniques for detecting viral DNAs are based on the ability of the unique sequence of nucleotides in a DNA strand to bond with (`hybridize`) to a sequence complementary to it. Thus, armed with a DNA sequence for all or a unique part of a papillomavirus type, it is possible to use this as a `homing probe` in order to detect the virus in a sample of cervical cells from a patient. The DNA for use as probe is labelled either with a radioactive isotope or nonradioactive label so that it can be detected later. To increase the sensitivity of the test we have utilized a method for amplification of the HPV DNA sequences in the sample.
Background to approach used to amplify viral DNA
In order to increase the sensitivity of the detection technique we use a method described originally for diagnosis of genetic diseases. This is known as a `polymerase chain reaction` (PCR). It is used to amplify enzymatically a specific DNA sequence before hybridization with synthetic oligonucleotide probe. A typical amplification factor is .about.250,000 copies starting from one copy of viral DNA. Such an approach not only increases sensitivity, but fulfills requirements of specificity, speed, simplicity, and amenability to nonradioactive detection methods expected of a more versatile testing procedure. It is also amenable to assembly as a kit and to automation, both of which we have accomplished. The PCR technique is described in papers that deal with prenatal diagnostic testing for specific genetic abnormalities [Sakii et al., Science 230: 1350-4, 1985; Sakii et al., Nature 324: 163-6, 1986; Scharf et al., Science 233: 1076-8, 1986]. The PCR technique is the subject of the following: Australian Patent Application AU-A-55322/86, Cetus Corp." Process for Amplifying Nucleic Acid Sequences", U.S. Priority Date 28.3.85; Australian Patent Application AU-A-55323/86, "Amplification and Detection of Target Nucleic Acid by Hybridization Probe", U.S. Priority Date 28.3.85.
Briefly, a small, unique portion of the HPV DNA sequence, .about.100-200 bp long, is amplified by the PCR procedure. In the examples a region in the E6 region has been chosen. However, any other region in the viral DNA may also be chosen. The PCR step requires two .about.20 mer oligonucleotide primers that flank the region to be amplified. One primer is complementary to the (+)-strand of a region of the DNA and the other is complementary to the (-)-strand. The annealing of primer to the (+)-strand of denatured sample viral DNA, followed by extension with, e.g., the Klenow fragment of Escherichia coli DNA polymerase, or other enzymes that carry out a similar reaction, and deoxynucleotide triphosphates results in the synthesis of a (-)-strand fragment containing a `target` sequence residing between the hybridization sites of the primer. At the same time a similar reaction occurs with the other primer, creating a new (+)-strand. The principle of the method is shown in the following diagram. ##STR1##
Since these newly-synthesized DNA strands are themselves templates for the PCR primers, repeated cycles of denaturation, primer annealing, and extension result in the exponential accumulation of the .about.100-200 bp region defined by the primers. Next, the specific DNA is detected. Various means are possible for doing this. The amount of DNA produced may be sufficient for direct visualization after electrophoresis on a gel and staining.